The present invention pertains to the dissociation and removal of highly hydrated macroscopic volumes of proteinaceous tissue; more particularly, the present invention pertains to the dissociation and removal of highly hydrated macroscopic volumes of proteinaceous tissue using rapid variable direction energy field flow fractionization.
The present invention is described in terms of vitreoretinal surgery; however, those of ordinary skill in the art will understand the applicability of this invention to medical procedures in other areas in the body of humans or animals.
For decades, prior art procedures for vitreoretinal posterior surgery have relied on mechanical or traction methods for: 1) tissue removal with shear cutting probes (utilizing either a reciprocating or rotary cutter); 2) membrane transection using scissors, a blade, or vitreous cutters; 3) membrane peeling with forceps and picks; and 4) membrane separation with forceps and viscous fluids. While improvements in mechanisms, materials, quality, manufacturability, system support, and efficacy have progressed, significant advancements in posterior intraocular surgical outcomes are primarily attributable to the knowledge, fortitude, skill, and dexterity of the operating ophthalmic physicians.
Traction-free removal of intraocular tissue during vitreoretinal surgery is nearly impossible with the current arsenal of mechanical medical instruments. Through the application of skill, precise movement, experience, and knowledge, operating physicians have been able to minimize the traction from the use of mechanical medical instruments during tissue removal but are unable to eliminate it. Mechanical or traction surgical methods utilize a shearing action to sever tissue bonds. This shearing action inherently puts tension on the tissue to be removed, that tension, in turn, is transferred to the retinal membrane. Because of the use of mechanical or traction surgical methods, the forces which impart motion to the cutting element of the mechanical medical devices being used to sever tissue bonds are superimposed on the retinal membrane. Despite the skill and the care of the ophthalmic surgeon, this superimposition of the forces associated with traction surgical methods onto the retinal membrane gives rise to the possibility of damage to the retinal membrane.
A potential traction-free surgical method that has been used in generating conformational changes in protein components involves the application of high intensity pulsed electrical fields; however, the use of a high-intensity pulsed electrical field has not made its way into delicate surgical procedures such as vitreoretinal surgery.
High-intensity pulsed electric fields have found numerous applications in the medical field, the food industry, and in the machining of micromechanical devices. Examples of medical field use include delivery of chemotherapeutic drugs into tumor cells, gene therapy, transdermal drug delivery, and bacterial decontamination of water and liquid foods. In the food industry, high-intensity ultrashort-pulsed electric fields have found commercial use in sterilization and decontamination. Finally, the machining and surface modification techniques used for Micro Electric Mechanical Systems (MEMS) chips employ high-intensity ultrashort-pulsed electrical fields.
Manipulation of biological structures, such as macromolecules, cellular membranes, intracellular organelles, and extracellular entities, has been the focus of recent research by both biophysics and biochemical engineering groups. Under the general heading of electrokinetics, the response of biological tissues to electric fields has been used in research, diagnostic, and therapeutic applications.
Non-Surgical Electrokinetic Research and Development
Basic understanding of the invention described herein is best obtained through an appreciation of some of the prior-art nonsurgical technologies now in use for biochemical molecular research, therapeutic pharmaceutical developments, sterilization techniques, commercial polymerization, plasma research, and MEMS (lab-on-a-chip) advancements. Key aspects of these prior-art technologies are described below to demonstrate other systems in which proteinaceous material has been manipulated and compromised by the delivery of high-intensity pulsed electrical fields.
Electrorheology
Electrorheology (ER) is a phenomenon in which the rheology of fluids, to include biological fluids, is modified by the imposition of electrical fields (usually low DC fields). The electrical field imposed on the fluid induces a bulk-phase transition in the fluid with the strength of the electrical field being the most important parameter, and the frequency of the electrical field generally being the least important parameter. Most colloidal ER fluids demonstrate an increase in viscoelastic effects with increased field amplitude. Interestingly, a decrease in viscoelasticity of the fluid appears at the highest field strengths, but definitive research into the effect of field strength on viscoelasticity of the fluid is lacking, and the mechanism of ER remains unknown.
Electrophoresis
Electrophoresis (or dielectrophoresis) involves the movement of particles in an electrical field toward one or another electric pole, anode, or cathode. The electrophoresis process is used to separate and purify biomolecules (e.g., DNA and RNA separation). For materials that are on the order of nanometers to micrometers, the electrophoresis process works well for both highly specific isolation of materials and determination of material properties. During electrophoresis, electrical field induced phase transition in a confined suspension is the subject of a spatially uniform AC electrical field. This electrical-field-induced phase transition follows the well-known field-induced formation of a columnar structure in a suspension. When subjected to an external electrical field, the particles within the electrical field align themselves along the field direction, forming chains and columns. The chains and columns of particles are then stretched by the actions of the electrical field and fluid flow. The time for separation and isolation of particles is on the order of minutes to hours and often involves the application of multiple secondary processes. An ionic surfactant (e.g., sodium dodecyl sulfate SDS) and sample dilution are often used to enhance macromolecular separation. Ionic surfactants have the ability to form a chemical bridge between hydrophobic and hydrophilic environments, thus disrupting or diminishing the hydrophobic connecting forces needed to maintain native protein structure.
Field Flow Fractionation
Field Flow Fractionation (FFF) is a laboratory solution separation method comparable in many ways to liquid chromatography. In general, both the materials and size range of materials separated in FFF systems are complimentary to those analyzed using electrophoresis and liquid chromatography. In FFF systems, the separation protagonist (electrical field) is applied in a direction perpendicular to the direction of separation and creates spatial and temporal separation of the sample components at the output of the FFF channel. Separation in an FFF channel is based on differences in the retention (time) of the sample components. In turn, the retention in FFF systems is a function of the differences in the physiochemical properties of the sample, the strength and mode of the applied assault, and the fluid velocity profile in the separation channel. Utilization of FFF has reduced electrophoresis times from hours to minutes.
Electric Field Flow Fractionation
Arising from the work being done in machining Micro Electric Mechanical Systems (MEMS) is Electric Field Flow Fractionation (EFFF). EFFF is a process for the ex-vivo separation of nanoparticles, proteins, and macromolecules entrained in microchannels by applying electrical fields either in the axial or in the lateral direction. This technique is currently under study in connection with MEMS microphoresis devices. The method is based on axial flow of analyte under the action of an electrical potential (unidirectional lateral electrical field). The separation performance and the retention time of particulate samples in the flow channel depend on the interaction of the sample with the electrical field applied transverse to the flow field in the channel. Dissociation of protein complexes, disruption of protein connections, and subsequent fractionization has been achieved with EFFF. An increase in retention, resulting in much better separation, has also been seen with the application of periodic (oscillating) electrical fields in EFFF.
In addition, the application of pulsed potentials with alternating polarity has been shown to increase the effectiveness of the electrical field. It has been postulated that shear plays a significant role in chain scission, since local deformation of proteinaceous tissue in any electrical field gradient is pure elongation. Quantified by a strain rate and axes of extension and compression, careful manipulation of array geometry and flow-field strength can result in significant extension of the majority of the macromolecules. Microchips have been designed that can generate rotational, extensional, and shear electrical field patterns, as long as the input voltages are changed. Separation time on a 1.25 cm chip has been reduced to approximately 5 seconds.
Electroporation
Electroporation is another nonsurgical prior-art technology that has been used to reversibly and transiently increase the permeabilization of a cell membrane. Introduced in about 1994, electroporation (EP) to enhance the delivery of drugs and genes across cell membranes in-vitro has become a standard procedure in molecular biology laboratories in the last decade. Electroporation is a technique in which pulses of electrical energy, measured in kilovolts per centimeter, having a duration in the microsecond-to-millisecond range, cause a temporary loss of the semi-permeability of cell membranes. This temporary loss of the semi-permeability of cell membranes leads to ion leakage, escape of metabolites, and increased cellular uptake of drugs, molecular probes, and DNA. Some prior-art applications of electroporation include introduction of plasmids or foreign DNA into living cells for transfection, fusion of cells to prepare hybridomas, and insertion of proteins into cell membranes. Classically, pulse durations in the order of 0.1 to 10 milliseconds and electrical field strength of kV/cm, depending on cell type and suspension media, have been utilized. The mechanism of electroporation (i.e., the opening and closing of cellular channels) is not completely understood.
Adaptations of the electroporation technology have been used for drug delivery. U.S. Pat. No. 5,869,326 and Published U.S. Patent Application 2004/0176716 both describe instruments for transcutaneous drug delivery. Published U.S. Patent Application 2004/021966 describes a catheter instrument for intravascular delivery of therapeutic drugs and in-vitro drug delivery using electrode array arrangements. U.S. Pat. No. 6,653,114 teaches a means for electrode switching. U.S. Pat. No. 6,773,736 and U.S. Pat. No. 6,746,613 have adapted electroporation technology to decontaminate products and fluids by causing cell deactivation and death. U.S. Pat. No. 6,795,728 uses electroporation-induced cell death as the basis for an apparatus and method for reducing subcutaneous fat deposits in-vivo.
Nanosecond Pulsed Electrical Field
Nanosecond Pulsed Electrical Field (nsPEF) technology is an extension of electroporation technology described above, to include in-vivo application, where a square or trapezoidal pulse formed with significantly shorter duration (1-300 ns), together with considerably higher electric fields (up to 300 kV/cm), is utilized. nsPEF evolved from advances in pulse-power technology. The use of this pulse-power technology has lead to the application of nanosecond-pulsed electronic fields (nsPEF) with field intensities several hundred times higher than the pulses of electrical energy used in electroporation to cells and tissues without causing biologically significant temperature increases in the samples tested. Using very few pulses of electrical energy, the effects of nsPEF are essentially non-thermal. In contrast to classical electroporation techniques, the effects of nsPEF on mammalian cells have only recently been explored. Application of nsPEF of appropriate amplitude and duration creates transient cellular permeability increases, cellular or subcellular damage, or even apoptosis. In in-vivo nanosecond electroporation, the goal is to obtain an even distribution of an efficacious electrical field within a narrow time window.
Current research has shown that the application of nanosecond pulses (kV/cm) to tissues can energize electrons without heating ions or neutral particles. It has been found that an ultrashort-pulsed energy field (Electromagnetic EM, Laser, or High Intensity Focused Ultrasound HIFU) can be used to temporarily and reversibly increase the permeability of cell membranes or even compromise intracellular components without affecting the cell membrane. It has also been found that higher energies will excite ions and may cause the formation of short-lived radicals (OH and O2+). This finding has lead to the development of processes for sterilization and decontamination whereby cells are killed. The use of still higher energies may cause the formation of super-charged plasma arcs which attack cellular bonds at the molecular level.
Electro-Osmosis
Electro-osmosis (EO) is a technique used to transport or mix fluid for use in micro devices. A key concept is to exploit different charging mechanisms and polarization strength of the double layer at the electrode/electrolyte interface, to produce a unidirectional Maxwell force on the fluid, which force generates through-flow pumping. In “induced-charge electro-osmosis” (ICEO), an effect is created which produces microvortices within a fluid to enhance mixing in microfluidic devices. Mixing can be greatly enhanced in the laminar flow regime by subjecting the fluid to chaotic-flow kinematics. By changing the polarity and the applied voltage, the strength and direction of the radial electro-osmotic flow can be controlled.
Other Electrokinetic Phenomena
Electrokinetic phenomena are not limited to that described above. Recent variants associated with very large voltages and unique electrical fields in MEMS research have demonstrated interesting and counter-intuitive effects occurring with variable applied electrical fields, including the finding that the electrophoretic mobility of colloids is sensitive to the distribution of charges, rather than simply the total net charge.
Tissue Removal
All of the processes described above are applicable to manipulation of macromolecules, but not to the extraction or removal of macroscopic volumes of proteinaceous tissue by tissue dissociation. As other systems using pulsed energy with tissues employ high levels of energy, it has been found that higher energies delivered through the use of longer pulse durations, pulse trains, repetition rates, and exposure times will cause thermal effects or the formation of super-charged plasma. These thermal effects or the formation of super-charged plasma have been effectively utilized in several devices to develop surgical instruments for tissue cutting. In these instruments, a microsize (thickness or projection) plasma region is created about an instrument. Within the super-charged plasma are charged electrons, ions, and molecules with an erratic motion which, when contacted with tissues or cells, attack bonds at the molecular level—thereby ablating or obliterating via sublimation the target tissue or tissue surface. The formation of super-charged plasma relies on electron avalanche processes—high rate of tunneling by electrons from the valence band to the continuum to form electron plasma avalanche ionization. The density of this super-charged plasma rapidly builds up by virtue of additional tunneling as well as field-driven collisions between free electrons and molecules. A major goal of the treatment of tissue with super-charged plasma is nondestructive surgery; that is, controlled, high-precision removal of diseased sections with minimum damage to nondiseased tissue. The size and shape of the active plasma are controlled by probe design, dimensions, and media. Both gaseous and fluid media have been employed. Within a liquid, an explosive vapor may be formed.
Pulsed Electron Avalanche Knife
The Pulsed Electron Avalanche Knife (PEAK) disclosed in Published U.S. Patent Application 2004/0236321 is described as a tractionless cold-cutting device. A high electrical field (nsPEF 1 to 8 kV, 150 to 670 uJ) is applied between an exposed microelectrode and a partially insulated electrode. The application of this high electrical field leads to a plasma formation manifested in the form of micrometer-length plasma streamers. It is the size of the exposed electrode which controls the dimensions of the plasma streamers. The plasma streamers, in turn, create an explosive evaporation of water on a micron scale. Pulsed energy is critical. Precise, safe, and cost-effective tissue cutting has been demonstrated. Even with the electrode scaled down in size to the micron level, the plasma discharges must be confined to the probe tip, because ionization and explosive evaporation of liquid medium can disrupt the adjacent tissue and result in cavitation bubble formation. The high pressure achieved during plasma formation, the fast expansion of vapor bubble (>100 m/sec), and the subsequent collapse of the cavity that can extend the zone of interaction is mainly mechanical due to rapid bubble vapor cool down. In ophthalmic surgery, the volatility and aggressiveness of the effect caused by the use of a PEAK could be detrimental to retinal integrity.
Coblation
Coblation, or “Cold Ablation,” uses radio frequency RF in a bipolar mode with a conductive solution, such as saline, to generate plasma which, when brought into contact with a target tissue, sublimates the surface layer of the target tissue. The range of accelerated charged particles is short and is confined to the plasma boundary layer about the probe and to the surface of tissue contact. Coblation energizes the ions in a saline-conductive solution to form a small plasma field. The plasma has enough energy to break the tissue's molecular bonds, creating an ablative path. The thermal effect of this process has been reported to be approximately 45-85° C. Classically, RF electrosurgical devices use heat to modify tissue structure. The generation of a radio frequency induced plasma field, however, is viewed as a “cold” process, since the influence of the plasma is constrained to the plasma proper, and the plasma layer maintained is microscopically thin. The plasma is comprised of highly ionized particles of sufficient energy to achieve molecular dissociation of the molecular bonds. The energy needed to break the carbon-carbon and carbon-nitrogen bonds is on the order of 3-4 eV. It is estimated that the Coblation technique supplies about 8 eV. Due to the bipolar configuration of the electrodes and the impedance differential between the tissue and the saline solution, most of the current passes through the conductive medium located between the electrodes, resulting in minimal current penetration into the tissue and minimal thermal injury to the tissue. If the threshold of energy required to create plasma is not reached, current flows through the conductive medium and the tissue. Energy absorbed by both the tissue and the conductive medium are dissipated as heat. When the threshold of energy needed to create plasma is reached, impedance to RF current flow changes from almost purely resistive-type impedance into a more capacitive-type impedance. Similar to the drawbacks of the PEAK for ophthalmic surgery, the use of coblation techniques may be too aggressive for surgical applications near the retina.
Plasma Needle
The plasma needle is yet another device that allows specific cell removal or rearrangement without influencing surrounding tissue. Use of the plasma needle is a very exacting technique which utilizes a microsize needle affixed to a hand-operated tool to create a small plasma discharge. An electrical field is created between the needle tip and a proximal electrode with an inert gas (helium) flowing there between. The small plasma discharge contains electrons, ions, and radicals—with the ions and radicals controllable by the introduction of a contaminant, such as air, into the inert gas. It has been postulated that the small size of the plasma source (plasma needle) creates ROS (reactive oxygen species) and UV light emissions at such minute levels as to alter cell function or cell adhesion without damaging the cells themselves. However, an increase in ROS (i.e., air) in the inert gas along with an increased irradiation time can lead to cell death. While shown to exert an influence across thin liquid layers, use of the plasma needle is not optimal in a total liquid environment, as often found in ophthalmic surgery.
Spark Erosion
Spark erosion technology is a cousin to the plasma technologies discussed above. The spark erosion device utilizes a pulsed energy field of 250 kHz, 10 ms duration, and up to 1.2 kV to produce a vapor. As the electric breakdown of vapor occurs, a small spark (<1 mm) is formed. With up to a 1.7 mm far-field effect, the cutting performance from spark erosion is similar to electrosurgery, but, like plasma—only the plasma contacts tissue.
Lasers
Lasers represent another traction-free technology that has been used to break down tissue macro molecules. Lasers have been utilized in ophthalmic surgery since about 1960. The greatest success in laser usage has been in the area of non-invasive retinal coagulation in diseases such as diabetic retinopathy, central vein occlusion, and choroidal neovascularization in age-related macular degeneration or ischemic retinal vasculitis. Lasers have also been used extensively in anterior eye applications for such applications as corneal sculpting and glaucoma. Attempts to utilize lasers in posterior ophthalmic surgeries have achieved mixed results. Non-invasive (trans-corneal/lens or trans-sclera) techniques are not practical, due to the absorptive properties of these intervening tissues. The extraordinary precision needed in intraocular surgery of the retina and vitreous requires the use of increasingly refined invasive techniques for tissue manipulation and removal. The tissue/laser interaction regimes include 1) thermal—conversion of electromagnetic energy into thermal energy; 2) photochemical—intrinsic (endogenous) or injected (exogenous) photosensitive chemicals (chromophores), activated by absorption of laser photos; 3) photoablative—direct photodissociation of intramolecular bonds of absorption of photons; and 4) electromechanical—thermionic emission or multiphoton production of free electrons leading to dielectric breakdown and plasma production. It has been found that lasers are costly and require the use of shields and backstops on uniquely designed laser probes to protect fine intraocular tissues from stray laser energy and far-field thermal effects. However, recent developments in femtosecond-pulsed lasers have opened new possibilities in fine surgical applications.
Other Tissue Removal Methods
Methods currently employed to disrupt intraocular tissues include morcellation (fragmentation), which is the objective of mechanically shearing vitrectomy devices; liquefaction as accomplished by thermal (protein denaturizing) or enzymatic reactions; and sublimation via laser or plasma treatments. Sublimation via laser or plasma treatments actually compromises bonds on a molecular level, whereas morcellation and liquefaction affect the binding mechanism of lesser strength (i.e., non-covalent bonds).
Accordingly, despite many advances in vitreoretinal surgery, a need still remains for an effective apparatus and method for the dissociation and removal of highly hydrated macroscopic volumes of protein tissues, such as vitreous and intraocular tissues, during vitreoretinal surgery.